1. Technical Field
This invention relates to a method of high sensitivity analysis through chemiluminescence measurement in use of enzymatic reactions, antigen-antibody reactions and nucleic acid hybridization in the areas of clinical laboratory testing, food inspection, environmental analysis, inspection of animals and plants, and manufacturing process control checking.
2. Background Art
The luminescent reaction using oxidation of luminol, isoluminol or a derivative thereof [abbreviated as chemiluminescent DPD (2,3-dihydro-1,4-phthalazinedione) in the following] by a peroxidase is used for immunoassay, analysis of elastase, analysis of glucose and analysis of oxidants. It is known that for improving the luminescent intensity of said luminescent reaction, it is effective to add an enhancer, such as those shown below, to the reaction system.
(1) 6-Hydroxybenzothiazole (Patent Publication No. SHO 56-5000252) PA0 (2) A certain kind of phenol having a substitution group (Patent Publication No. SHO 59-171839) PA0 (3) A certain kind of aromatic amine (Patent Publication No. SHO 61-54453)
However, such enhancers have the following difficulties.
The enhancer (1) is generally of lower luminescence intensity and smaller signal-to-background ratio.
The typical enhancer of the class (2) is p-iodophenol. Its luminescent signal is high, but the background is also high, therefore the signal-to-background ratio is relatively low.
In the case of the typical enhancer of the class (3) or N,N,N',N'-tetramethylbenzidine, the rise to the luminescent peak is slow, and much time is required for measurement.
For the reaction mechanism of the enhancer effect, there are reports proposing the requirement of efficient formation of luminol semiquinone radical [Thorpe, G. H. C. and Kricka, L. J., "Bioluminescence and Chemiluminescence: New Perspectives," Scholmerich, J., Andreesn, R., Kapp, A., Ernst, M. and Woods, W. G. (Eds), John Wiley, Chichester, pp. 199-208 (1987)] and the requirement of efficient formation of phenoxy radical [Hodgson, M. and Jones, P., Journal of Bioluminescence and Chemiluminescence, Vol. 3, pp. 21-25 (1989)].
However, the phenol derivatives include a number of compounds which do not show an enhancer effect, and it is difficult to choose an effective enhancer based upon the foregoing theories.
Also, for detection or quantification of a gene in a virulent microorganism, a prostaglandin or any other physiologically active substance, luteinizing hormone (LH) and other anterior pituitary hormones, and cytokines such as interleukin in blood, it is required to make a determination based on very small amount in body fluid. Thus, a high sensitivity detection system was called for, and for improvement of the detection sensitivity, an enhancer of higher efficiency has been desired.